Reduction of 11, 17-keto steroids by trichomonads



United States Patent REDUCTION OF 11,17-KETO STEROIDS BY TRICHOMONA-DSOldrieh K. Sebelr, Kalamazoo Township, Kalamazoo No Drawing. ApplicationSeptember 17, 1956 Serial No. 610,399

12 Claims. (Cl. 195-51) The present invention relates to a novel methodfor the production of certain 17,8-hydroxy steroids, and is moreparticularly concerned with the reduction of 17-ketosteroids of theandrostane, etiocholane and estrane series by animal microorganisms aswell as such reduction with simultaneous dehydrogenation of thesaturated 1,2-bond by the same animal microorganism in the presence ofbacteria living in commensalism with the animal microorganism.

It is already known how to reduce 17-ketosteroids by chemical means,such as by hydrogen in its nascent state (e. g., sodium metal inethanol, Fernholz, U. S. Patent 2,356,154) or by reduction with ametallic hydride, e. g. lithium aluminum hydride [Herr et al. I. Am..Chem. Soc. 75 5927 (1953)]. However in these reductions of l-7-ketogroups it is necessary to protect desired keto groups which occur inother positions to avoid simultaneous reductions of the desired ketogroups. This can be accomplished by means of known carbonyl groupreacting agents for example by forming ketals, enamines, semicarbazonesor the like. Thus, the knownchemical reductions necessitate at least twoadditional steps: formation of the protective group and a subsequentremoval thereof. Since the ll-keto group is generally difficult toprotect it does not react with most of the carbonyl group reactingagents, this group is often also reduced necessitating a subsequentoxidation while the 1'7-hydroxy group is protected by an ester group.Thus to produce ll-ketotestosterone from adrenosterone Ecf. Herr, J. Am.Chem. Soc. 75, 5927 (1953)] six steps are required. In contrast, in theinstant invention ll-ketotestost'erone is produced from adrenosterone inone step.

The reduction of l7-ketosteroid's with plant microorganisms (yeast) isknown; for example, Herzog et al., I. Am. Chem. Soc. 75, 266 (1953)showed conversion of adrencsterone by yeast to ll-ketotestosterone in 62percent yield and Ma'moli and Vercellone, Ber. 70, 470' (1937) describedreduction of androstene-dione to testosterone byyeast.

A disadvantage of the use of plant organism is the fact that theyinvariably split ester groups Whenever present in the steroid. Thus fromestrone esters Mamoli, Ber. 71, 2696 (1938) obtained free estradiol.Free estrone was not reduced at all by yeast. It has now been found thatanimal organisms of the family Trichomonadidae, especially of the generaTrichomonas, Tritrichomoms and Pentatrichomonas and in particular thespecies T richomonas gallinae and Tritrichomonas foetus are useful forthe selective reduction of the l7keto group of l7-keto steroids toproduce stereospecifically the corresponding 17,8-hydroxysteroid.

According to the classification system of Kudo, Protozoology, Charles C.Thomas Co., Springfield, Illinos, 1946, the family Trichomonadidaebelongs to the suborder Monornonadina, which belongs to the orderPolymastigina of subclass Zoomastigina, of class Mastigophora, subphylumPlasmodroma and phylum Protozoa. Other authors, e. g., Banner B. Morganand Philip Hawkins,

2,877,161 Patented Mar. 10, 1959 Veterinary Protozoology, BurgessPublishing 00., Minneapolis, Min, 1952, classify the familyTrichomonadidae as part of the order Trichomonadida. In thisspecification the nomenclature of Kudo will be used.

It is an object of the instant invention to provide a new process forthe reduction of 17-keto groups and 17- keto-steroids such. as17-ketoandrostanes, l7-ketoestranes and 17-keto'etiocholanes. It is alsoan object of the present invention to provide such a process ofreduction of a'17-keto group of l7-ketosteroids with simultaneousdehydrogenation of a saturated bond in the 1,2-position of the steroid,to form an unsaturated double bond between the carbon atoms 1' and 2, bythe simultaneous action of protozoa of the family Trichomonadidae in thepresence of a plant organism of. the genus Corynebacterium. Otherobjects will be apparent to those skilled in the art to which-thisinvention pertains.

The present invention provides an easy method to reduce the 17-ketogroup in l7-ketosteroids to the. corresponding 17,8-hydroxyl group andthereby produce a more active compound such as testosterone fromandrostenedione, ll-ketotestosterone from adrenosterone, estradiol fromestrone and the like. The instant combination of Trichomonas andCorynebacterium is also useful to produce dehydrogenation at the1,2-carbon atoms simultaneously with the reduction of the 11-keto groupdue to the unique fact that the Trichomonas can exist in commensalismwith Corynebacterium under more or less anerobic conditions. Forexample, Tricho-monas in mix.- tures with Corynebacterium, in a one-stepoperation, converted 4-androstene-3,l 7-dione to l-dehydrotestosterone,adreno'sterone to l-dehydro-ll-ketotestosterone and 11B- hydroxy-nor-4androstene-3,l7-dione to llfi-hydroxyestradiol, which compounds haveincreased androgenic and anabolic activity or increasedanabolic-androgenic ratio, or are otherwise more desirable such as, forinstance, estradiol which is used more than the 17-keto analogue,est'rone.

The starting materials of the instant reaction are 17- ketosteroids,such as compounds belonging to the 17- keto-androstane class includingthose 17-ketoandrostanes having double bonds for example in the-4,5, inthe 5,6 or in the 1,2-position such as, for example, 4-androstene-3,1'7-dion'e, 1,4-androstadiene-3,17-dlone, epi-d'ehydroandrosterone,11p-hydroxyAnndrostene-B,l7-dione, adrenosterone, androstane-3,17-dione,11u-hydroxy-4-androstene- 3,17-dione, Bfi-acetoxy-S-androsten-17-one;17-ketoetio cholanes, such as etiocholane'-3',1'7-dione,3a-hydrox'yetiocholan-17-one, 3a-acetoxy-etiocholan-l7-one, 38-acetoxyetiocholan-17-one; 17-keto-estrane compounds including thosel7-ketoestranes' having double bonds in positions 1, 3, 5 or 4 such asestIane-3,17-dione, 4-estrene-3,l7 dione, 1118 hydroxy 4 cstrene 3,17dione, 4 estrene- 3', l1,l7'-trione (noradrenosterone), estrone, or thelike.

The method of the present invention, in its broader aspects, consists insubjecting a starting compound having a l7'-keto group such asthestarting compounds named above to the reducing action of anerobicallygrown genera of the family Trichomonadidae, especially the generaTrichomonas, Tritrichomonas, and Pentatrichomonas.- Among the speciesuseful. in this conversion are the known Trich0m nas gallinae,Trichomoms elongata (buccalis), Trichomonas vaginalis, Trichomonaslinearis, Trichomonas termitis, Trichomonas anseri, and the like;Tritrichomonas foetus, T ritvri'chomonas brevicollis, T ritrichom'onaseberthi, Tritrichomonas fecalis and the like; and Penlatrichomonasgallinarium, Pentatrich m0nas hominis, with Trichomonas gallinae andTritrichomonas foetus preferred.

If in addition to the reduction of a 17-keto group, dehydrogenation atthe 1,2-carbon atoms is desired to form a l-dehydro-17-hydr0xysteroid,belonging to the androstane, estrane or etiocholane classes, theselected Trichomonas, Tritrichomonas or Pentatrichomonas species isgrown anaerobically or nearly anaerobically in the presence ofCorynebacterium such as C. equi ATCC 10146, C. simplex ATCC 6946, C.pseudodiphtheriticum (hotfmanii) ATCC 6981 or C. renale ATCC 10848 andthe like. In the preferred embodiment of this aspect of the presentinvention, whenever simultaneous reduction of the l7-keto group anddehydrogenation of the steroid on the 1,2-position is desired, thecombination of T richomonas gallinae or Trichomonas foetus withCorynebacterium simplex or Corynebacterium equi is used.

The trichomonads are grown under conditions well known in the art [cf.,for example, Andr Lwoff, Biochemistry and Physiology of Protozoa, volumeI, Academic Press Inc., Publishers, New York, 1951, pages 150 to 174;Banner B. Morgan, Veterinary Protozoology, Burgress Publishing Company,Minneapolis, Minn., revised edition 1952].

In regard to the medium or menstruum for the growth of Trichomonas, itis necessary to provide basic food materials in easily assimilable form,such as assimilable nitrogen-containing nutrients, e. g., proteins, beefbroth (peptonized), liver extracts and liver-infusion extractions, heartinfusion, pancreatic digest of casein, aminoacids, lysine, asparagine,sterile blood serum, and defibrinated blood, gelatin, eggs in dispersedform, agar, guanine, xanthine, horse serum, and the like; assimilablecarbon compounds, e. g., carbohydrates, e. g., glucose, lactose,maltose, fructose, sucrose, and other sugars, dextrin, soluble starches,ground potato, wheat, corn, and the like, and soluble glycogen, and thelike.

As mineral constituents, the medium may contain, naturally present oradded, available calcium, sodium, potassium, copper, cobalt, iron,gallium, magnesium, manganese, arsenic, molybdenum, vanadium, and othermetals. Sulfur may be provided by the addition of cystine, alkalithioglycollate, thiamine, biotin, or the like; phosphorus by theaddition of soluble phosphates, potassium acid phosphate, as lecithin,cephalin, ovovitelline, phytine, or the like, boron and iodine in traceswhen desirable.

For the growth of Trichomonas, necessary vitamins should desirably beadded, such as ascorbic acid (vitamin C). Other vitamins of importancefor optimum growth comprise vitamin B and B pyridoxine, vitamin A and D.Cholesterol is desirable for several Trichomonas species, especially T.gallinae and T. foetus. Other desirable growth factors, often added,include: folic acid, glutamic acid, linolenic acid, u-tocopherol,calcium pantothenate, a-carotene, riboflavin, choline, estradiol,ergosterol, and the like.

Nutritional differences between species of Trichomonas are quitepronounced as is known; thus, while Trichomonas gallinae and T. foetuscan utilize such sugars as galactose, fructose, lactose, Trichomonasvaginalis is unable to assimilate these sugars.

The nutrient solution should also contain buifer substances suitable forthe required growth conditions of the selected Trichomonas species. Thusaccording to Lwotf (loc cit.) Trichomonas gallinae are most viable at apH of about 7 (e. g., with a calcium carbonate buffer present in themedium) and die at a pH of about 4.5 which is reached after five to sixdays of cultivation in an unbuffered medium. Best pH for Trichomonasvaginalis is between 5.4 to six, according to the same author.

Similarly for optimum growth conditions of Trichomonas, it is necessaryto regulate the temperature closely to the one best suitable for theselected species. Most species and strains are viable and multiplybetween 32 to forty degrees centigrade, with an optimum temperature forTrichomonas gallinae, T. vaginalis and T. colubro'rum of 37 degreescentigrade, for Trich monas foetus, Wittes strain V, Riedmiillers strain78 and Glasers strain G between 32 to 37 degrees centigrade while Wittesstrain IV has an optimal temperature of only 28 degrees (Lwoff loc.cit). Thus, the optimum temperature conditions for most species in vitroare either near or below the average body temperature of warm blooded(mammal or bird) hosts, seldom above the temperature of the host.

In the instant invention the medium which has been successfully usedconsisted of pancreatic digest of casein, cystine, yeast extract, sodiumthioglycollate, agar, horse serum, and glucose. When Corynebacterium wasused in conjunction with Trichomonas the Corynebacterium and theTrichomonas were simultaneously inoculated in the nutrient medium.Corynebacterium, especially C. equi ATCC 10146 and C. simplex ATCC 6946,increases the life-time of the trichomonad and allows simultaneously atwo to five-fold increase in the population number of Trichomonas pervolume unit. Since the trichomonads are cultivated in the absence of airand are also cultivated in the absence of air when Corynebacterium waspresent, it was surprising to find that the Corynebacterium in theabsence of air can produce a dehydrogenation which may be considered anoxidation reaction, while at the same time the Trichomonas producesreduction of 17-keto groups.

The general procedure used in the instant invention comprisesinoculating a prepared medium with Trichomonas and allowing anincubation time sufficient to raise a population between two to fourmillion per milliliter. This is usually obtained during a period of oneto two days. The culture is grown at a temperature suitable for optimumgrowth, for example, about 37 degrees centigrade for Trichomonas foetusand T richomonazs gallinae. However, temperatures between 32 and fortydegrees; centigrade may be used for the cultivation of the T richomonasspecies. After the population has grown to a number of two-four millionper milliliter the selected steroid is added, previously dissolved in anappropriate solvent, non-toxic to the microorganism, for example,propylene glycol, aqueous ethanol or the like. The concentration of thesolvent in the medium should be low, preferably in the neighborhood ofone percent to avoid the growth inhibition of the Trichomonas. Thesteroid is allowed to remain in contact with the selected Trichomonasspecies for generally one day. Occasionally the reaction time may beextended or diminished as deemed necessary for the particular reactionand thus may range from twelve to 72 hours. After the reaction isterminated, the contents of the flask are sterilized, as by killing theorganisms present with steam heat. Subsequently the total fermentationmaterial is extracted with water-immiscible solvents such as methylenechloride, ethylene dichloride, chloroform, carbon tetrachloride,

benzene, hexanes, toluene, ether, or the like. The steroids in theextracts are recovered by conventional means such as evaporation of thesolvent and purified as by recrystallization and chromatography, asdeemed necessary. If a more exhaustive recovery is desired, thefollowing procedure can be followed: after heating the fermentationmixture, the mixture is filtered to separate the beer from the solids.The beer is extracted with water-immiscible solvents as mentioned above,and the solids, derived from the food provided and the destroyedmicroorganisms, washed and extracted separately with acetone,Skellysolve B-hexanes, methylene chloride, chloroform, or otherconvenient solvents. The thus obtained extractives are then combinedwith the extractives from the beer, and together are evaporated andtreated as shown before to isolate and purify the steroids.

If the reaction is run in the presence of a selected Corynebacteriumsuch as Corynebacterium equi or Corynebacterium simplex, the procedureis the same as shown above. The Corynebacterium and Trichomonas areinoculated and cultivated together under the same conditions asdescribed.

The followingexamples are illustrative of the process and products ofthe present invention, but are not to be construed as limiting.

EXAMPLE 1 Testosterone from 4-andr0stene-3J7-dione A medium was preparedcontaining per one liter of solution fifteen grams of pancreatic digestof casein, 0.5 gram of cystine, five grams of yeast extract, five gramsof dextrose, 2.5 grams of sodium chloride, 0.5 gram of sodiumthioglycollate, 0.75 gram of agar and five per cent of horse serum. 100milliliters of this medium was then incubated with Trichomonas gallinaeat 37 degrees for a period of 24 hours after which time the populationreached between two and four million animals per milliliter. To theinoculated medium, thus obtained, a sterile solution of4-androstene-3,17-dione, sterilized under fifteen pounds of steampressure per square inch for twenty minutes was added. The addition wasmade by dissolving ten milligrams of the 4-androstene-3,l7-dione in 0.5milliliter of propylene glycol. The material was aseptically pipettedand the incubation continued at 37 degrees for another 24 hour period.Thereafter, the flask containing the fermentation mixture was heated ona steam bath for fifteen to twenty minutes, the beer separated from thesolid material by filtration and the beer extracted with three25-milliliter portions of methylene chloride and the solids extractedthree times with ten-milliliter portions of hot acetone. The extractswere combined, the solvent evaporated, the residue redissolved inacetone and filtered through a paper filter, evaporated to dryness andrecrystallized three times from methanol to give 6.5

milligrams of testosterone.

In the same manner as shown above, 800 milligrams of4-androstene-3,l7-dione were dissolved in twenty milliliters ofpropylene glycol and added to four liters of a medium prepared as above.The material was allowed to react in the fermentation solution for aperiod of 24 'hours and thereafter the mixture was sterilized by steamFraction Solvent Skellysolve B hexanes.

Skellysolve B hexane acetone 98: 2. Skellysolve B hexanezacetone 97:3.Skellysolve B hexane:acetone 96:4. Skellysolve B liexanezacetone 96:4.Skellysolve B hexanezacetone 95:5. Skellysolve B hexane:acetone 94:6.Skellysolve Bhexanmacetone 93:7. Skellysolve B hexanezacetone 93:7.Skellysolve B hexanmacetone 90:10. Acetone 100%.

Fractions 5 through 13 were combined, evaporated and the materialrecrystallized from methanol to give 730 milligrams of testosterone.

EXAMPLE 2 II-ketotestosterone from adrenosterone In the same manner asshown in Example 1, 800 milligrams of adrenosterone were treated withTrichomonas gallinae in the same type of medium to give 755 milligramsof ll-ketotestosterone.

EXAMPLE 3 In the same manner as shown in Example 1, but usingTritrichomonas foetus instead of T richomonas gallinae,

adrenosterone was converted in 95 percent yield by papergram analysis toll-ketotestosterone.

EXAMPLE 4 I-dehydr ostestosterone In the same manner as given in Example1, 1,4.-andros- .tadiene 3,17-dione was converted with Trichomonas galrJinae in 87 percent yield to l-dehydrotestosterone.

Using Pentatrichomonas hominis, .Pentatrichomonas gallinarumor-Trichomonas vaginalis instead of Tricho- .monas gallinae alsoproduced l-dehydrotestosteronefrom 1,4-androstadiene-3,17-dione.

EXAMPLE 5 1 .lfiv'hydroxytestosterone In the same manner given inExample 1, l1 B-hydroxy- 4-androstene-3,17-dione was converted tollfi-hydroxytestosterone in 86 percent yield with Trichomonas gallinae.

EXAMPLE 6 318-acetoxy-1 7 fl-hy droxy -5 -an drostene In the same mannergiven in Example 1, 3B-acetoxy- 5-androstene-17-one was reacted withTrichomonas gal- Zinae to give in 7l percent yield3fi-acetoXy-17B-hydroxy- S-androstene. This conversion is distinctly.difierent from any similar reduction by a plant organism (yeast), sincethe plant organism in almost percent efficiency hydrolyze ester groupspresent in steroids.

EXAMPLE 7 1 1 a,1 7 fl-dihydr0xy-4-andr0stene-3mne .In the same manneras given in Example 1, Ila-hydroxy-4-androstene-3,17-dione was convertedwith Irivchomomzs gallinae to l1a,17fi-dihydroxy-4-androsten 3- one inninety percent yield. I

In the same manner but using Pentatrichomonas gallinarum forTrichomonats gallinae l1a,l7fi-dihydroXy-4- androsten-3-one was obtainedfrom 11a-hydroxy-4-androstene-3,17-dione.

EXAMPLE 8 In the same manner as given invExample l,3fi-hydroxy-5-androsten-17-one was converted to3fi,17fl-dihydroxy-S-androstene with T richomonas foetusxin 86 .percentyield.

EXAMPLE 9 3 0a,] 7 5-41 ihydroxy-etiocholane In the same manner asgivenin Example 1, 3a-hydroxy-etiocholan-17-one was subjected tofermentation with Trichomonas vaginalis to give 3o ,l73-dihydroxyetiocholane with folliculoid activity.

EXAMPLE 1O Estradiol from estrone EXAMPLE 1 1 1 -dehydr-1 1-ketotestoster0ne from adrenosterone In 100 milliliters of a mediumconsisting of 1.5 grams of casein, 0.5 gram of yeast extract, 0.5 gramof dextrose, 0.25 gram of sodium chloride, 0.05 gram of sodiumthioglycollate, 0.075 gram of agar, 0.05 gram of cystine and containingfive percent of horse serum Corynebucterium simplex ATCC 6946 andTrichomonas gallinae were grown at 37 degrees for aperiod of 24 hours.After that time, ten milligrams of 4-androstene-3,l1,17-trione(adrenosterone), dissolved in one milliliter of propylene glycol, wasadded and the incubation continued at 37 degrees for one additional dayto give l-dehydro-ll-ketotestosterone.

EXAMPLE 12 1 -dehydr0test0ster0ne In the same manner given in Example11, 4-androstene- 3,17-dione was converted with Corynebacterium equiATCC 10146 and Trichomonas foetus to give l-dehydrotestosterone, whichwas isolated by extraction from the fermentation mixture with methylenechloride and purified by chromatography with acetone and Skellysolve Bhexane as shown in Example 1.

EXAMPLE 13 1 lfi-hydroxyestradiol from 1 1 fi-hydroxyl-estrene- 3,1 7 -diorze In the same manner as given in Example 11, Corynebacterium equiand Trichomonus gallinae were grown together for a period of 36 hoursand thereafter the steroid, 11fi-hydroxy-4-estrene-3,l7-dione was added.The mixture was incubated for another period of 36 hours, thereuponheated under steam bath and extracted with methylene chloride as shownin Example 1 to give 1 lfi-hydroxyestradiol.

EXAMPLE 14 1 1 -ketoestradi0l In the same manner given in Example 11,Corynebacterium equi and T richomonas foetus were grown together for aperiod of 36 hours Whereafter noradrenosterone.(4-estrene-3,l1,17-trione) was added and the mixture grown for a periodof 48 hours. The mixture was then .heated on steam bath for a period ofthirty minutes and extracted with methylene chloride to give afterevaporation of the extract ll-ketoestradiol.

EXAMPLE 15 1 -dehydro-1 1 B-Izydroxytestosterone In the same mannergiven in Example 11, Coryuebacterium simplex and Trichomonas gallirraewere grown together for a period of 24 hours and thereafter 11,8-hydroxy-4-androstene-3,17-dione was added to give 1-dehydro-lIB-hydroxytestosterone, which was isolated and purified asshown in Example 1.

In the same manner as given in Examples 11 through 15, other1-dehydro-17 8-hydroxy steroids can be produced from the corresponding17-ketosteroids by simultaneous fermentation of the starting17-ketosteroid with a species of Corynebacterium and a species selectedfrom the family Trichomonadidae. Preferred are the combinations ofCorynebacterium simplex and Corynebacterium equi With T richomonusgallinue and Tritrichomonas foetus. Representative compounds, thusproduced, include l-dehydro-l7-hydroxyetiocholan-3-one frometiocholane-3,l7-dione, 1,5-androstadiene-3fl,17,3-diol fromepi-dehydroandrosterone, 1 dehydro-175-hydroxyandrostan-3-one fromandrostane-3,17-dione, l-dehydro-lluhydroxytestosterone from1la-hydroxy-4-androstene,17- dione, lla-hydroxy-estradiol from1la-hydroxy-4-estrene- 3,17-dione, and the like.

limited to the exact de ails of operation or exact compounds shown anddescribed, as obvious modifications and equivalents will be apparent toone skilled in the art, and the invention is therefore to be limitedonly by the scope of the appended claims.

We claim: 1

l. A process for the production of 17,8-hydroxy steroids whichcomprises: cultivating a species of the protozoan family Trichomonadidaeselected from the genera Tri chomonas, Tritrichomonas andPentatrichomonas in a. nutrient medium, in the presence of a17-ketosteroid selected from the group consisting of l7-ketoandrostane,17-ketoetiocholane and l7-ketoestrane to convert the 17- keto group ofsaid 17-keto steroid to a 17B-hydroxy group.

2. A process for the production of 17,8-hydroxy steroids whichcomprises: cultivating a species of the protozoan family Trichomonadidaeselected from the genera Trichomonas, Tritrichomonas andPentatrichomonas in a nutrient medium, containing assimilablenon-steroidal carbon, nitrogen, sulfur and phosphorus, and a17-ketosteroid selected from'the group consisting of 17-keto-.androstane, l7-ketoetiocholane and 17-ketoestrane to obtain thecorresponding 17p-hydroxy steroid and isolating the thus obtained17f3-hydroxy steroid.

3. A process for the production of 17 3-hydroxy steroids whichcomprises: cultivating T richomonas gallinue in a nutrient mediumcontaining assimilable nonsteroidal carbon, nitrogen, sulfur andphosphorus, and a l7-ketosteroid selected from the group consisting of17- ketoandrostane, l7-ketoetiocholane and 17-ketoestrane compounds toobtain the corresponding l7fi-hydroxy steroids and isolating the thusobtained 17fl-hydroxy steroids.

4. A process for the production of 17,3-hydroxy steroids whichcomprises: cultivating Tritrichomonas foetus in a nutrient mediumcontaining assimilable nonsteroidal carbon, nitrogen, sulfur andphosphorus, and a 17-ketosteroid selected from the group consisting of17- ketoandrostane, l7-ketoetiocholane and l7-ketoestrane compounds toobtain the corresponding 17B-hydroxy steroids and isolating the thusobtained 17fi-hydroxy steroids.

5. A process for the production of testosterone which comprises:cultivating a species of the family Trichomonadidae selected from thegenera Trichomonas, Tritrichomonas and Pentatrichomonas in a nutrientmedium containing assimilable non-steroidal carbon, nitrogen, sulfur andphosphorus, and 4-androstene-3,l7- dione to obtain testosterone andisolating the thus-prepared testosterone.

.6. A process for the production of testosterone which comprises:cultivating Trichomonas gallinae in a nutrient medium containingassimilable non-steroidal carbon, nitrogen, sulfur and phosphorus, and4-androstene-3,17- dione to obtain testosterone and isolating thethus-prepared testosterone.

7. A process for the production of ll-ketotestosterone which comprises:cultivating a species of the family Trichomonadidae selected from thegenera Trichomonas, Tritrichomonas and Pentatrichomonas in a nutrientmedium containing assimilable non-steroidal carbon, nitrogen, sulfur andphosphorus and adrenosterone to obtain ll-ketotestosterone and isolatingthe thus-prepared l l-ketotestosterone.

8. A process for the production of ll-ketotestosterone which comprises:cultivating Trichomonas gallinae in a nutrient medium containingassimilable non-steroidal carbon, nitrogen, sulfur and phosphorus, andadrenosterone to obtain ll-ketotestosterone and isolating thethusprepared ll-ketotestosterone.

9. A process for the production of l-dehydro-l7B-hydroxy steroids whichcomprises: cultivating a'species of the protozoan family Trichomonadidaeselected .from the genera Trichomonas, Tritrichomonas andPentatrichomonas and a Corynebacterium selected from the speciesCorynebacterium simplex and Corynebacterium equi, in a nutrient mediumcontaining assimilable nonsteroidal carbon, nitrogen, suatur andphosphorus, and a 17-ketosteroid, selected from the group of17-ketoandrostane, 17-ketoetiocholane and 17-ketoestrane to efiectsimultaneous dehydrogenation on the 1,2-position and reduction of the17-keto group to a 17,8-hydroxy group, and isolating the thus-obtainedcorresponding 1- dehydro-Ufi-hydroxy steroid.

10. A process for the production of l-dehydrotestosterone whichcomprises: cultivating Trichomonas foetus and Corynebacterium equi in anutrient medium containing assimilable non-steroidal carbon, nitrogen,sulfur and phosphorus, and 4-androstene-3,17-dione to effectsimultaneous dehydrogenation on the 1,2-position and reduction of the17-keto group to a l7fihydroxy group and isolating the thus producedl-dehydrotestosterone.

11. A process for the production of llfi-hydroxyestradiol whichcomprises: cultivating the protozoan T richomonas gallinae andCorynebacterium equi in a nutrient medium containing assimilablenon-steroidal carbon, nitrogen, sulfur and phosphorus, andllp-hydroxy-4-estrene-3,17-dione to effect simultaneous dehydrogenationon the 1,2-p0sition and reduction of the 17- keto group to a 17B-hydroxygroup and isolating the thus produced llfl-hydroxyestradiol.

12. A process for the production of l-dehydro-llketotestosterone whichcomprises: cultivating the protozoan Trichomonas gallinae andCorynebacterium simplex in a nutrient medium containing assimilablenon-steroidal carbon, nitrogen, sulfur and phosphorus, and adrenosteroneto effect simultaneous dehydrogenation on the 1,2- position andreduction of the 17-keto group to a 17shydroxy group and isolating thethus produced l-dehydro- 1 l-ketotestosteronc.

References Cited in the file of this patent Nobile et aL: Jour. Am.Chem. Soc. 77 (1955), p. 4184.

Vitamins and Hormones, XIV, Academic Press, 1956, pp. 391 to 394, 397.

1. A PROCESS FOR THE PRODUCTION OF 17$-HYDROXY STERIODS WHICH COMPRISES:CULTIVATING A SPECIES OF THE PROTOZOAN FAMILY TRICHOMONADIDAE SELECTEDFROM THE GENERA TRICHOMONAS, TRITRICHOMONAS AND PENTATRICHOMONAS IN ANUTRIENT MEDIUM, IN THE PRESENCE OF A 17-KETOSTEROID SELECTED FROM THEGROUP CONSISTING OF 17-KETOANDROSTANE, 17-KETOETIOCHOLANE AND17-KETOESTRANE TO CONVERT THE 17KETO GROUP OF SAID 17-KETO STEROID TO A17$-HYDROXYL GROUP.